[Fe]-Hydrogenase, Cofactor Biosynthesis and Engineering.

[Fe]-hydrogenase catalyzes the heterolytic cleavage of H2 and reversible hydride transfer to methenyl-tetrahydromethanopterin. The iron-guanylylpyridinol (FeGP) cofactor is the prosthetic group of this enzyme, in which mononuclear Fe(II) is ligated with a pyridinol and two CO ligands. The pyridinol ligand fixes the iron by an acyl carbon and a pyridinol nitrogen. Biosynthetic proteins for this cofactor are encoded in the hmd co-occurring (hcg) genes. The function of HcgB, HcgC, HcgD, HcgE, and HcgF was studied by using structure-to-function analysis, which is based on the crystal structure of the proteins and subsequent enzyme assays. Recently, we reported the catalytic properties of HcgA and HcgG, novel radical S-adenosyl methionine enzymes, by using an in vitro biosynthesis assay. Here, we review the properties of [Fe]-hydrogenase and the FeGP cofactor, and the biosynthesis of the FeGP cofactor. Finally, we discuss the expected engineering of [Fe]-hydrogenase and the FeGP cofactor.

Computer-aided discovery of a novel thermophilic laccase for low-density polyethylene degradation.

Polyethylene (PE) and industrial dyes are recalcitrant pollutants calling for the development of sustainable solutions for their degradation. Laccases have been explored for removal of contaminants and pollutants, including dye decolorization and plastic degradation. Here, a novel thermophilic laccase from PE-degrading Lysinibaccillus fusiformis (LfLAC3) was identified through a computer-aided and activity-based screening. Biochemical studies of LfLAC3 indicated its high robustness and catalytic promiscuity. Dye decolorization experiments showed that LfLAC3 was able to degrade all the tested dyes with decolorization percentage from 39% to 70% without the use of a mediator. LfLAC3 was also demonstrated to degrade low-density polyethylene (LDPE) films after eight weeks of incubation with either crude cell lysate or purified enzyme. The formation of a variety of functional groups was detected using Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Damage on the surfaces of PE films was observed via scanning electron microscopy (SEM). The potential catalytic mechanism of LfLAC3 was disclosed by structure and substrate-binding modes analysis. These findings demonstrated that LfLAC3 is a promiscuous enzyme that has promising potential for dye decolorization and PE degradation.

Engineering a Formate Dehydrogenase for NADPH Regeneration.

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD+ -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP+ -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD+ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (kcat /Km ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.

Engineered Glycosidase for Significantly Improved Production of Naturally Rare Vina-Ginsenoside R7.

Ginsenosides are the main bioactive ingredients in plants of the genus Panax. Vina-ginsenoside R7 (VG-R7) is one of the rare high-value ginsenosides with health benefits. The only reported method for preparing VG-R7 involves inefficient and low-yield isolation from highly valuable natural resources. Notoginsenoside Fc (NG-Fc) isolated in the leaves and stems of Panax notoginseng is a suitable substrate for the preparation of VG-R7 via specific hydrolysis of the outside xylose at the C-20 position. Here, we first screened putative enzymes belonging to the glycoside hydrolase (GH) families 1, 3, and 43 and found that KfGH01 can specifically hydrolyze the ß-d-xylopyranosyl-(1 → 6)-ß-d-glucopyranoside linkage of NG-Fc to form VG-R7. The I248F/Y410R variant of KfGH01 obtained by protein engineering displayed a kcat/KM value (305.3 min-1 mM-1) for the reaction enhanced by approximately 270-fold compared with wild-type KfGH01. A change in the shape of the substrate binding pockets in the mutant allows the substrate to sit closer to the catalytic residues which may explain the enhanced catalytic efficiency of the engineered enzyme. This study identifies the first glycosidase for bioconversion of a ginsenoside with more than four sugar units, and it will inspire efforts to investigate other promising enzymes to obtain valuable natural products.

The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor.

In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

Engineering Isopropanol Dehydrogenase for Efficient Regeneration of Nicotinamide Cofactors.

Isopropanol dehydrogenase (IPADH) is one of the most attractive options for nicotinamide cofactor regeneration due to its low cost and simple downstream processing. However, poor thermostability and strict cofactor dependency hinder its practical application for bioconversions. In this study, we simultaneously improved the thermostability (433-fold) and catalytic activity (3.3-fold) of IPADH from Brucella suis via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H) by 1.23 × 106-fold. When these variants were employed in three typical bioredox reactions to drive the synthesis of important chiral pharmaceutical building blocks, they outperformed the commonly used cofactor regeneration systems (glucose dehydrogenase [GDH], formate dehydrogenase [FDH], and lactate dehydrogenase [LDH]) with respect to efficiency of cofactor regeneration. Overall, our study provides two promising IPADH variants with complementary cofactor specificities that have great potential for wide applications. IMPORTANCE Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used. Isopropanol dehydrogenase (IPADH) is one of the most attractive biocatalysts for nicotinamide cofactor regeneration. However, poor thermostability and strict cofactor dependency hinder its practical application. In this work, the thermostability and catalytic activity of an IPADH were simultaneously improved via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H). The resultant variants show great potential for regeneration of nicotinamide cofactors, and the engineering strategy might serve as a useful approach for future engineering of other oxidoreductases.

Removing the Obstacle to (-)-Menthol Biosynthesis by Building a Microbial Cell Factory of (+)-cis-Isopulegone from (-)-Limonene.

Microbial synthesis of plant-based (-)-menthol is of great interest because of its high demand (≈30 kiloton per year) as well as unique odor and cooling characteristics. However, this remains a great challenge due to the yet unfilled gap between (-)-limonene and (+)-cis-isopulegone. Herein, the first artificial and effective system was developed for (+)-cis-isopulegone biosynthesis from (-)-limonene by recruiting two bacterial enzymes to replace their inefficient counterparts from Mentha piperita, limonene-3-hydroxylase, and isopiperitenol dehydrogenase. A cofactor self-regenerative recombinant Escherichia coli strain was constructed by introducing a formate dehydrogenase for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and an engineered microbial isopiperitenol dehydrogenase. The production of (+)-cis-isopulegone (up to 281.2 mg L-1 ) was improved by 36 times compared with that of the initial strain. This work lays a reliable foundation for the microbial synthesis of (-)-menthol.

Stereoselective synthesis of chiral δ-lactones via an engineered carbonyl reductase.

A carbonyl reductase variant, SmCRM5, from Serratia marcescens was obtained through structure-guided directed evolution. The variant showed improved specific activity (U mg-1) towards most of the 16 tested substrates and gave high stereoselectivities of up to 99% in the asymmetric synthesis of 13 γ-/δ-lactones. In particular, SmCRM5 showed a 13.8-fold higher specific activity towards the model substrate, i.e., 5-oxodecanoic acid, and gave (R)-δ-decalactone in 99% ee with a space-time yield (STY) of 301 g L-1 d-1. The preparative synthesis of six δ-lactones in high yields and with high enantiopurities showed the feasibility of the biocatalytic synthesis of these high-value-added chemicals, providing a cost-effective and green alternative to noble-metal catalysis.

Effect of first-stage aeration on treatment of domestic sewage in different hybrid constructed wetlands.

Two sets of hybrid constructed wetlands (HCWs) with the first-stage aeration were used to treat actual domestic sewage in this paper, where the effects of three important factors of aeration mode, hydraulic loading rates (HLR), and aeration volume on the removal of pollutants in both HCWs were studied in contrasts. In addition, the pollutant removal efficiency, the contribution of plants, and the characteristics of biofilm in both HCWs were explored. The results of 250-day experiment showed that the TN removal capacity of HCW combining vertical flow CW with horizontal flow CW (VF-HF) was better than HCW's converse combination (HF-VF) in treatingsewage, while the removal efficiency of COD and NH4+-N were similar, and the concentrations of TN and COD in the effluent of VF-HF could successfully meet the National discharge requirements. Compared with the continuous aeration, the intermittent aeration only had a little effect on the removal of COD and NH4+-N, but could improve TN removal performance in both HCWs. Meanwhile, increasing the aeration volume was beneficial to remove NH4+-N but not TN in HCWs. In addition, although the pollutant removal performances in both HCWs were impacted, the removal capacity of TN in VF-HF was only affected a little, when HLR was increased by 50%. The contribution of plants' uptake accounted for about 10% to nitrogen removal and 20% to phosphorus removal in both HCWs. The biomass at the filler surface near the plant rhizosphere was greater than that in the non-rhizosphere zones, and the impact of plant rhizosphere on the nitrification activity of biofilm was significantly greater than that on denitrification activity in both HCWs.

Multicenter Consistency Assessment of Valvular Flow Quantification With Automated Valve Tracking in 4D Flow CMR.

OBJECTIVES: This study determined: 1) the interobserver agreement; 2) valvular flow variation; and 3) which variables independently predicted the variation of valvular flow quantification from 4-dimensional (4D) flow cardiac magnetic resonance (CMR) with automated retrospective valve tracking at multiple sites. BACKGROUND: Automated retrospective valve tracking in 4D flow CMR allows consistent assessment of valvular flow through all intracardiac valves. However, due to the variance of CMR scanners and protocols, it remains uncertain if the published consistency holds for other clinical centers. METHODS: Seven sites each retrospectively or prospectively selected 20 subjects who underwent whole heart 4D flow CMR (64 patients and 76 healthy volunteers; aged 32 years [range 24 to 48 years], 47% men, from 2014 to 2020), which was acquired with locally used CMR scanners (scanners from 3 vendors; 2 1.5-T and 5 3-T scanners) and protocols. Automated retrospective valve tracking was locally performed at each site to quantify the valvular flow and repeated by 1 central site. Interobserver agreement was evaluated with intraclass correlation coefficients (ICCs). Net forward volume (NFV) consistency among the valves was evaluated by calculating the intervalvular variation. Multiple regression analysis was performed to assess the predicting effect of local CMR scanners and protocols on the intervalvular inconsistency. RESULTS: The interobserver analysis demonstrated strong-to-excellent agreement for NFV (ICC: 0.85 to 0.96) and moderate-to-excellent agreement for regurgitation fraction (ICC: 0.53 to 0.97) for all sites and valves. In addition, all observers established a low intervalvular variation (≤10.5%) in their analysis. The availability of 2 cine images per valve for valve tracking compared with 1 cine image predicted a decreasing variation in NFV among the 4 valves (beta = -1.3; p = 0.01). CONCLUSIONS: Independently of locally used CMR scanners and protocols, valvular flow quantification can be performed consistently with automated retrospective valve tracking in 4D flow CMR.

Engineering of an oleate hydratase for efficient C10-Functionalization of oleic acid.

Oleate hydratase catalyzes the hydration of unsaturated fatty acids, giving access to C10-functionalization of oleic acid. The resultant 10-hydroxystearic acid is a key material for the synthesis of many biomass-derived value-added products. Herein, we report the engineering of an oleate hydratase from Paracoccus aminophilus (PaOH) with significantly improved catalytic efficiency (from 33 s-1 mM-1 to 119 s-1 mM-1), as well as 3.4 times increased half-life at 30 °C. The structural mechanism regarding the impact of mutations on the improved catalytic activity and thermostability was elucidated with the aid of molecular dynamics simulation. The practical feasibility of the engineered PaOH variant F233L/F122L/T15 N was demonstrated through the pilot synthesis of 10-hydroxystearic acid and 10-oxostearic acid via an optimized multi-enzymatic cascade reaction, with space-time yields of 540 g L-1 day-1 and 160 g L-1 day-1, respectively.

A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis.

Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.

Secretory expression of cyclohexanone monooxygenase by methylotrophic yeast for efficient omeprazole sulfide bio-oxidation.

Prochiral pyrmetazole can be asymmetrically oxidized into (S)-omeprazole, a proton pump inhibitor that is used to treat gastroesophageal reflux, by an engineered cyclohexanone monooxygenase (CHMOAcineto-Mut) that has high stereoselectivity. CHMOAcineto-Mut is produced by heterologous expression in Escherichia coli, where it is expressed intracellularly. Thus, isolating this useful biocatalyst requires tedious cell disruption and subsequent purification, which hinders its use for industrial purposes. Here, we report the extracellular production of CHMOAcineto-Mut by a methylotrophic yeast, Pichia pastoris, for the first time. The recombinant CHMOAcineto-Mut expressed by P. pastoris showed a higher flavin occupation rate than that produced by E. coli, and this was accompanied by a 3.2-fold increase in catalytic efficiency. At a cell density of 150 g/L cell dry weight, we achieved a recombinant CHMOAcineto-Mut production rate of 1,700 U/L, representing approximately 85% of the total protein secreted into the fermentation broth. By directly employing the pH adjusted supernatant as a biocatalyst, we were able to almost completely transform 10 g/L of pyrmetazole into the corresponding (S)-sulfoxide, with > 99% enantiomeric excess.

Structure-guided engineering of Pseudomonas dacunhael-aspartate ß-decarboxylase for l-homophenylalanine synthesis.

Structure-guided engineering of Pseudomonas dacunhael-aspartate ß-decarboxylase (AspBDC) resulted in a double mutant (R37A/T382G) with remarkable 15 400-fold improvement in specific activity reaching 216 mU mg-1, towards the target substrate 3(R)-benzyl-l-aspartate. A novel strategy for enzymatic synthesis of l-homophenylalanine was developed by using the variant as a biocatalyst affording 75% product yield within 12 h. Our results underscore the potential of engineered AspBDC for the biocatalytic synthesis of pharmaceutically relevant and value added unnatural l-amino acids.

Apical hypertrophic cardiomyopathy with left ventricular apical aneurysm: prevalence, cardiac magnetic resonance characteristics, and prognosis.

AIMS: Hypertrophic cardiomyopathy (HCM) with left ventricular apical aneurysm (LVAA) is associated with an increased risk of adverse cardiovascular events. However, the clinical significance of LVAA in apical HCM (ApHCM) has not been reported. This study aimed to investigate the prevalence, cardiac magnetic resonance (CMR) characteristics, and prognosis of LVAA in ApHCM patients. METHODS AND RESULTS: A total of 1332 consecutive ApHCM patients confirmed by CMR in our hospital were retrospectively analysed. LVAAs were identified in 31 patients of all ApHCM patients (2.3%, 31/1332). Besides, 31 age- and gender-matched ApHCM patients without LVAA were used for comparison. Of the 31 aneurysm patients (mean age, 53.8 ± 15.1 years old), 28 (90.3%) had clinical symptoms, and 3 (9.7%) had a family history of HCM. The rate of missed diagnosis of echocardiography for detecting LVAA was 64.5% (20/31), most (90%, 18/20) of unidentified LVAAs by echocardiography were small aneurysms (<20 mm). Compared with ApHCM patients without LVAA, the proportion of systolic mid-cavity obstruction and late gadolinium enhancement (LGE) presence, and the LGE extent in ApHCM patients with LVAA were significantly higher (all P<0.05). The Kaplan-Meier curves showed that the event-free survival rate in ApHCM patients with LVAA was significantly lower than that in ApHCM patients without LVAA (log rank, P = 0.010). CONCLUSION: ApHCM with LVAA is a very rare condition, which is often missed by echocardiography and could be reliably detected with CMR and is associated with a higher risk of adverse cardiovascular events compared with ApHCM without LVAA.

Research and application status of ecological floating bed in eutrophic landscape water restoration.

Ecological floating bed (EFB) has become the preferred technology due to its reputation of green economy, convenience, and efficiency in treating eutrophic landscape water. Based on the statistical analysis of abundant literatures, this paper systematically summarizes the component elements, design parameters, purification mechanism, purification ability, strengthening methods and the correlations among various parameters of EFB, and points out some issues existing in the current research and applications. The results show that the coverage of 5% ~ 38% and water depth of 60 ~ 110 cm should be recommended for EFB design. The microbial transformation-sedimentation contributes mostly to the removal of pollutant, leading to the contribution rate of 51.9% ± 26.4% to nitrogen (N) removal and 50.8% ± 20.4% to phosphorus (P) removal in water respectively. Meanwhile, the average purification abilities of EFB for carbon (C), N and P in water are 4.59 ± 3.82, 0.43 ± 0.35 and 0.04 ± 0.04 g m-2 d-1 respectively. The purification effect is relatively superior when the initial concentration of C, N and P in water is close to C: N: P = 115: 11: 1. In order to enhance the EFB purification efficiency, the methods of artificial aeration, biological chain extension, functional filler introduction, and composite EFB construction can be used. Furthermore, the purification ability of EFB per unit area is correlated positively with water temperature and initial pollutant concentration (r ≥ 0.577, p < 0.01), and correlated negatively with EFB coverage (r ≤ -0.598, p < 0.01). The future research of EFB should focus on enhancing its purification efficiency and seasonal adaptability, studying the mechanism of algae inhibition by allelochemicals, and exploring the harvesting management and resource utilization of plants. This paper provides more reasonable design parameters, feasible management strategies and prospective research directions for environmental managers and researchers who would like to adopt EFB to purify eutrophic landscape water.

[Strengthening Effect of Different Cattail Pretreatment Methods on the Denitrification of Horizontal Subsurface Flow in a Constructed Wetland].

Nitrogen removal in constructed wetlands (CWs) is highly affected by the supply of organic carbon. Thus, to enhance nitrogen removal in the horizontal subsurface flow of CWs, plant carbon sources were added during the downstream portion of the wetland. Moreover, the characteristics of static release and the denitrification potential of Typha were evaluated using three different pretreatment methods (i.e., minced, acid-heated, and alkali-heated). The average concentrations of COD released and the nitrate removal rate with the alkali-heated, acid-heated, and minced Typha were 89.57 mg·L-1 and 75.2%, 67.27 mg·L-1 and 67.2%, and 54.45 mg·L-1 and 23.5%, respectively. The results showed that different pretreatment methods resulted in the release of different amounts of acetic acid, and the alkali-heated Typha performed much better than the other pretreatments. Therefore, the alkali-heated Typha was selected and added to the middle of the horizontal subsurface flow CW, which improved the nitrogen-removal rate significantly; the average removal rate of TN and TP was higher than that of a control CW by 30.3% and 33.9%, respectively. However, the COD concentration of the CW with the alkali-heated Typha was not significantly increased.

Direct Access to Medium-Chain α,ω-Dicarboxylic Acids by Using a Baeyer-Villiger Monooxygenase of Abnormal Regioselectivity.

Baeyer-Villiger monooxygenases (BVMOs) are versatile biocatalysts in organic synthesis that can generate esters or lactones by inserting a single oxygen atom adjacent to a carbonyl moiety. The regioselectivity of BVMOs is essential in determining the ratio of two regioisomers for converting asymmetric ketones. Herein, we report a novel BVMO from Pseudomonas aeruginosa (PaBVMO); this has been exploited for the direct synthesis of medium-chain α,ω-dicarboxylic acids through a Baeyer-Villiger oxidation-hydrolysis cascade. PaBVMO displayed the highest abnormal regioselectivity toward a variety of long-chain aliphatic keto acids (C16 -C20 ) to date, affording dicarboxylic monoesters with a ratio of up to 95 %. Upon chemical hydrolysis, α,ω-dicarboxylic acids and fatty alcohols are readily obtained without further treatment; this significantly reduces the synthetic steps of α,ω-dicarboxylic acids from renewable oils and fats.

Discovery of Two Native Baeyer-Villiger Monooxygenases for Asymmetric Synthesis of Bulky Chiral Sulfoxides.

Two Baeyer-Villiger monooxygenases (BVMOs), designated BoBVMO and AmBVMO, were discovered from Bradyrhizobium oligotrophicum and Aeromicrobium marinum, respectively. Both monooxygenases displayed novel features for catalyzing the asymmetric sulfoxidation of bulky and pharmaceutically relevant thioethers. Evolutionary relationship and sequence analysis revealed that the two BVMOs belong to the family of typical type I BVMOs and the subtype ethionamide monooxygenase. Both BVMOs are active toward medium- and long-chain aliphatic ketones as well as various thioether substrates but are ineffective toward cyclohexanone, aromatic ketones, and other typical BVMO substrates. BoBVMO and AmBVMO showed the highest activities (0.117 and 0.025 U/mg protein, respectively) toward thioanisole among the tested substrates. Furthermore, these BVMOs exhibited distinct activity and excellent stereoselectivity toward bulky and prochiral prazole thioethers, which is a unique feature of this family of BVMOs. No native enzyme has been reported for the asymmetric sulfoxidation of bulky prazole thioethers into chiral sulfoxides. The identification of BoBVMO and AmBVMO provides an important scaffold for discovering enzymes capable of asymmetrically oxidizing bulky thioether substrates by genome mining.IMPORTANCE Baeyer-Villiger monooxygenases (BVMOs) are valuable enzyme catalysts that are an alternative to the chemical Baeyer-Villiger oxidation reaction. Although BVMOs display broad substrate ranges, no native enzymes were reported to have activity toward the asymmetric oxidation of bulky prazole-like thioether substrates. Herein, we report the discovery of two type I BVMOs from Bradyrhizobium oligotrophicum (BoBVMO) and Aeromicrobium marinum (AmBVMO) which are able to catalyze the asymmetric sulfoxidation of bulky prazole thioethers (proton pump inhibitors [PPIs], a group of drugs whose main action is a pronounced and long-lasting reduction of gastric acid production). Efficient catalysis of omeprazole oxidation by BoBVMO was developed, indicating that this enzyme is a promising biocatalyst for the synthesis of bulky and pharmaceutically relevant chiral sulfoxide drugs. These results demonstrate that the newly identified enzymes are suitable templates for the discovery of more and better thioether-converting BVMOs.